The human central nervous system is composed of 100 billion neurons interconnected into precise circuits by 100 trillion synaptic connections. These circuits are required for nervous system functions including perception, thought and behavior. Much is known about the early steps in circuit formation in which neurites extend to target regions containing the correct synaptic partners. Much less is known about how individual neurons choose the correct synaptic partner when they reach a target region with many neurons. We have developed a novel method to visualize contact and synaptogenesis between specific neurons in vivo. We propose to use this method and take advantage of the simple, well-characterized nervous system of C. elegans to elucidate molecular mechanisms that underlie synaptic partner choice. This is an important area of study, as altered synaptogenesis is thought to play a role in disorders such as schizophrenia and autism. This proposal is relevant to the NIGMS mission to support research that increases understanding of life processes including the field of developmental biology, that lay the foundation for advances in disease diagnosis, treatment, and prevention, and to train the next generation of scientists. To understand how correct synaptic partner choice is mediated, we have developed a genetically encoded fluorescent trans-synaptic marker to visually label synaptic contacts between individual neurons of interest in complex environments called NLG-1 GRASP, for Neuroligin-1-mediated GFP Reconstitution Across Synaptic Partners. We have also labeled pre- and postsynaptic neurites with the red mCherry fluorophore. Together, these markers enable us to instantly assess correct synaptic partner choice by visualizing neurite contact and synaptogenesis between pre- and postsynaptic neurons of interest in live animals, making it feasible to use genetic methods to discover genes mediating this fundamental process. In addition, we have developed these markers in C. elegans, the only model organism for which there is a complete synaptic map, making it ideal for the study of synaptic partner choice. Using this marker, we have found that two proteins previously studied for their role in cell migration and axon guidance in other systems, UNC-40/DCC (deleted in colorectal cancer) and UNC-6/Netrin, have a novel role in mediating synaptic partner choice between sensory neurons and interneurons in the C. elegans ventral nerve cord. Our research will further characterize this role and define the pathway(s) that mediate synaptic partner choice. Our specific aims are: 1) to characterize the role of the UNC-40/DCC receptor and the UNC-6/Netrin ligand in synaptic partner choice, 2) to investigate genes that transduce UNC-40/DCC-mediated axon guidance and cell migration signals for roles in synaptic partner choice, and 3) to identify new genes that mediate synaptic partner choice by conducting a forward genetic screen. Understanding the mechanisms that regulate circuit formation will bring us closer to understanding and treating neurological diseases. PUBLIC HEALTH RELEVANCE: For the nervous system to function correctly, neurons must faithfully identify cellular partners with which to form synapses. Altered synapse formation is thought to underlie neurological diseases, such as schizophrenia and autism. We seek to identify the molecular mechanisms that underlie synaptic partner choice, as understanding these mechanisms will bring us closer to understanding and treating neurological diseases.